Biofilms are surface-attached microbial communities which are usually formed by microorganisms when they are exposed to unfavorable conditions for survival. Biofilm formation mechanisms are the subject of current investigations, however, yet biofilm formation and composition are not fully understood due to its quite complicated formation dynamics and highly heterogeneous composition including extracellular DNA, proteins and carbohydrates present in the biofilm matrix. As a densely populated community, bacteria are protected from unfavorable conditions within the complex structure of a biofilm. Because of the protective role of the biofilm matrix and bacterial heterogeneity in particular with respect to physiological adaptations, it is impossible to completely eradicate biofilms even after exposure to the highest deliverable doses of antibiotics.
Also, there is no convenient diagnostic tools to identify the presence of biofilms in a host organism. One available technique is to use fluorescence in situ hybridization techniques but, this can only be done by means of biopsies from tissue that are suspected to contain bacterial biofilms. Molecular tools that enables elucidation biofilm formation mechanisms and the identification—visualization of already formed biofilms are very important for academic purposes and clinical applications.
According to the National Institute of Health, more than 80% of all infections are associated with biofilms [Immunology of Biofilms (PA-07-288); NIH, Department of Health and Human Services, 2007-01-09]. Furthermore, biofilm bacteria are able to disperse and spread to new areas [Research on Microbial Biofilms (PA-03-047); NIH, National Heart, Lung, and Blood Institute, 2002-12-20]. Bacteria in biofilm-associated infections often appear non-cultureable [Li, L.; Mendis, N.; Trigui, H.; Oliver, J. D.; Faucher, S. P. Front. Microbiol. 2014, 5, 258]. Therefore, rapid and reliable identification of biofilm-associated infections are important for choosing the proper treatment strategy in the clinics, such as antimicrobial administration or surgical removal of the infected tissue.
The biofilm matrix (in which the bacteria are embedded) consists of extracellular polymeric substances (EPS) including DNA, proteins and polysaccharides. Several compounds have been reported to specifically label EPS components. For example, DNA staining dyes such as ethidium bromide, Syto9 or DAPI are frequently used to localize extracellular DNA in biofilms [Trachoo, N. Songklanakarin J. Sci. Technol. 2003, 25, 807]. Hippeastrum Hybrid (Amaryllis) Lectin, HHA, that specifically binds to either 1,3- or 1,6-linked mannosyl units in polysaccharides is used for biofilm detection after conjugating with a fluorophore due to its binding specificity with the Psl polysaccharide, a key components of P. aeruginosa biofilms [Ma, L.; Lu, H.; Sprinkle, A.; Parsek, M. R.; Wozniak, D. J. J. Bacteriol. 2007, 189, 8353].
Recently, ligand targeted ultrasound contrast agents (UCAs) were reported to detect biofilm in vitro under high-frequency scanning acoustic miscroscopy [Anastasiadis, P.; Mojica, K. D.; Allen, J. S.; Matter, M. L. J. Nanobiotechnol. 2014, 12, 24]. Even though it showed the possibility as a biofilm detecting tool of application, there is no established method for non-destructive in vivo biofilm detection by imaging so far.